This application claims priority from Great Britain Application GB 9908410.5, filed Apr. 13, 1999.
This invention relates to certain pyridine derivatives useful as urokinase inhibitors, and in particular to 2-diaminomethyleneaminopyridine derivatives, alternatively named as 2-pyridylguanidine derivatives, useful as urokinase inhibitors.
Urokinase (urinary-type plasminogen activator or uPA; International Union of Biochemistry classification number EC.3.4.21.31) is a serine protease produced by a large variety of cell types (smooth muscle cells, fibroblasts, endothelial cells, macrophages and tumour cells). It has been implicated as playing a key role in cellular invasion and tissue remodelling. A principal substrate for uPA is plasminogen which is converted by cell surface-bound uPA to yield the serine protease plasmin. Locally produced high plasmin concentrations mediate cell invasion by breaking down the extracellular matrix. Important processes involving cellular invasion and tissue remodelling include wound repair, bone remodelling, angiogenesis, tumour invasiveness and spread of metastases.
Beneficial effects of urokinase inhibitors have been reported using anti-urokinase monoclonal antibodies and certain other known urokinase inhibitors. For instance, anti-urokinase monoclonal antibodies have been reported to block tumour cell invasiveness in vitro (W. Hollas, et al, Cancer Res. 51:3690; A.Meissauer, et al, Exp. Cell Res. 192:453 (1991); tumour metastases and invasion in vivo (L. Ossowski, J. Cell Biol. 107:2437 (1988)); L. Ossowski, et al, Cancer Res. 51:274 (1991)) and angiogenesis in vivo (J. A. Jerdan et al, J. Cell Biol. 115[3 Pt 2]:402a (1991). Also, Amiloride(trademark), a known urokinase inhibitor of only moderate potency, has been reported to inhibit tumour metastasis in vivo (J. A. Kellen et al, Anticancer Res., 8:1373 (1988)) and angiogenesis/capillary network formation in vitro (M. A. Alliegro et al, J. Cell Biol. 115[3 Pt 2]:402a). Urokinase activity has also been implicated as a factor in psoriasis : Jensen and Lavker (1996) Cell Growth Diff 7, 1793-1804 Baker B S and Fry L (1992). Br J Dermatol, 126(1), 1-9.2; Spiers E M, et al (1994). J Invest Dermatol, 102(3), 333-338.3. Grondahl-Hansen J, et al (1987). J Invest Dermatol. 88(1), 28-32. Gissler H, et al (1993). Br J Dermatol, 128(6), 612-8; Venning Va., et al (1993). Clin Exp Dematol, 18(2), 119-23.
Conditions of particular interest for treatment by urokinase inhibitors include chronic dermal ulcers (including venous ulcers, diabetic ulcers and pressure sores), which are a major cause of morbidity in the ageing population and cause a significant economic burden on healthcare systems. Chronic dermal ulcers are characterised by excessive uncontrolled proteolytic degradation resulting in ulcer extension, loss of functional matrix molecules (e.g. fibronectin) and retardation of epithelisation and ulcer healing. A number of groups have investigated the enzymes responsible for the excessive degradation in the wound environment, and the role of plasminogen activators has been highlighted (M. C. Stacey et al., Br. J Surgery, 80, 596; M. Palolahti et al., Exp. Dermatol., 2, 29, 1993; A. A. Rogers et al., Wound Repair and Regen., 3, 273, 1995). Normal human skin demonstrates low levels of plasminogen activators which are localised to blood vessels and identified as tissue type plasminogen activator (tPA). In marked contrast, chronic ulcers demonstrate high levels of urokinase type plasminogen activator (uPA) localised diffusely throughout the ulcer periphery and the lesion, and readily detectable in wound fluids.
uPA could affect wound healing in several ways. Plasmin, produced by activation of plasminogen, can produce breakdown of extracellular matrix by both indirect (via activation of matrix metalloproteases) and direct means. Plasmin has been shown to degrade several extracellular matrix components, including gelatin, fibronectin, proteoglycan core proteins as well as its major substrate, fibrin. Whilst activation of matrix metalloproteases (MMPs) can be performed by a number of inflammatory cell proteases (e.g. elastase and cathepsin G), the uPA/plasmin cascade has been implicated in the activation of MMPs in situ, providing a broad capacity for degrading all components of the extracellular matrix. Furthermore, in addition to its effect on production of plasmin, uPA has been shown to catalyse direct cleavage of fibronectin yielding antiproliferative peptides. Thus, over-expression of uPA in the wound environment has the potential to promote uncontrolled matrix degradation and inhibition of tissue repair. Inhibitors of the enzyme thus have the potential to promote healing of chronic wounds.
Several related enzymes such as tPA, which also acts via production of plasmin, play a key role in the fibrinolytic cascade. Because of this it is desirable that a uPA inhibitor has adequate potency and selectivity for uPA relative to both tPA and plasmin to avoid the possibility of anti-fibrinolytic side effects.
The utility of such potent and selective urokinase inhibitors is highlighted by the broad range of invasive biological processes mediated by urokinase. These processes include, but are not limited to, wound healing, angiogenesis-dependent conditions such as retinopathy, bone restructuring, embryo implantation in the uterus, infiltration of immune cells into inflammatory sites, ovulation, spermatogenesis, tissue remodelling during wound repair and organ differentiation, fibrosis, local invasion of tumours into adjacent areas, metastatic spread of tumour cells from primary to secondary sites, and tissue destruction in arthritis.
Various aromatic amidines have been reported to inhibit uPA (J. D. Geratz, M. C.-F. Cheng, Thromb. Diathes. haemorrh. (Stuttg.), ;3 230, 1975; J. Stxc3xcirzebecher, F. Markwardt, Pharmazie, 33, 599, 1978; J. D. Geratz et al., Thromb. Res., 24, 73, 1981). The compounds reported in these publications are generally relatively weak and/or non-selective for uPA relative to other related serine proteases. EP 0 568 289 A2 discloses a series of benzo[b]thiophene-2-carboxamidines with significantly greater potency and selectivity with respect to tPA and plasmin (see also M. J. Towle et al., Cancer Res., 53, 2553, 1993; A. J. Bridges et al., Bioorg. Med. Chem., 1, 403, 1993).
There are few reports of guanidine derivatives as uPA inhibitors. Amiloride(trademark) (see below) is a weak but selective inhibitor of uPA (J.-D. Vassalli, D. Belin, FEBS Letters, 214, 187, 1987), and various 2-, 3- and 4-substituted phenylguanidines are reported to have a similar level of potency (H. Yang et al., J. Med. Chem., 33, 2956, 1990). 
M. Dukat et al, in J. Med Chem. 39, 4017 (1996) disclose various arylguanidines as a novel class of 5-HT3 ligands, the disclosure including the compound 2-guanidinopyridine. This compound is also disclosed by P J Taylor et al, in J. Chem.Soc.Perkin Trans. (II) 1765 (1986).
The substances described herein are potent reversibly-competitive inhibitors of urokinase enzymatic activity, with selectivity for urokinase relative to certain other important proteases, including the fibrinolytic enzymes tissue-type plasminogen activator (tPA) and plasmin.
The selectivity of the instantly-claimed substances for inhibition of urokinase over inhibition of other proteases such as tPA and plasmin, and the fact that they inhibit reversibly, prevents them from having thrombogenic properties. Thus, according to the present invention, there is provided a compound of formula (I): 
or a pharmaceutically acceptable salt thereof, or solvate of either entity,
wherein
R1 is H, halogen, CN, C1-6 alkyl optionally substituted by one or more halogen, or C1-6 alkoxy optionally substituted by one or more halogen,
R2 and R3 are each independently H, halogen, C1-6 alkyl optionally substituted by one or more halogen or C1-6 alkoxy, aryl, (Cn-alkylene)CO2H, (Cn-alkylene)CO2(C1-6 alkyl), (Cn-alkylene)CONR5R6, CHxe2x95x90CHR7, CHxe2x95x90CHCO2H, CHxe2x95x90CHCONR5R6, CHxe2x95x90CHSO2NR5R6, Cxe2x95x90CR7, O(Cm-alkylene)OH, O(Cm-alkylene)OR8, OR8, O(Cm-alkylene)CONR5R6, CH2OR8 or CH2NR5R6,
R4 is Nxe2x95x90C(NH2)2 or NHC(xe2x95x90NH)NH2,
R5 and R6 are each independently H, C1-6 alkyl optionally substituted by OH or CO2H, het(C1-6 alkylene) or aryl(C1-6 alkylene), or can be taken together with the nitrogen to which they are attached, to form a 4- to 7-membered saturated ring optionally containing an additional hetero-moiety selected from O, S or NR9,
and which ring is optionally benzo-fused,
and which optionally benzo-fused ring is optionally substituted by up to three substituents independently selected from OH, halogen, CO2H, CO2(C1-6 alkyl) and C1-6 alkyl,
R7 is C1-6 alkyl, aryl or het;
R8 is C1-6 alkyl, aryl, het, aryl(CHCO2H) or aryl(C1-6 alkylene);
R9 is H, C1-6 alkyl, or CO(C1-6 alkyl);
wherein xe2x80x9carylxe2x80x9dincluding the aryl moiety of the aryl(C1-6 alkylene) group, means phenyl optionally substituted by up to three substituents independently selected from halogen, C1-6 alkyl, (Cn-alkylene)CO2H, (Cn-alkylene)CO2(C1-6 alkyl), (Cn-alkylene)CN, C1-6 alkoxy, CN, (Cn-alkylene)CONR5R6, CHxe2x95x90CHCO2H, CHxe2x95x90CHCONR5R6, CHxe2x95x90CHSO2NR5R6, O(Cm-alkylene)OH, CH2NR5R6, and O(Cm-alkylene)CONR5R6;
xe2x80x9chetxe2x80x9d means an optionally benzo-fused 5- or 6-membered saturated or unsaturated heterocycle linked by any available atom in the heterocyclic or benzo-ring (if present), which heterocyclic group is selected from dioxolyl, furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, and pyranyl,
and which optionally benzo-fused heterocycle is optionally substituted by up to three substituents independently selected from halogen, C1-6 alkyl, (Cn-alkylene)CO2H, (Cn-alkylene)CO2(C1-6 alkyl), (Cn-alkylene)CN, (Cn-alkylene)CONR5R6, CHxe2x95x90CHCO2H, CHxe2x95x90CHCONR5R6, CHxe2x95x90CHSO2NR5R6, O(Cm-alkylene)OH, CH2NR5R6, and O(Cm-alkylene)CONR5R6;
n is 0, 1 or 2;
m is 1 or 2;
and wherein the xe2x80x9cC-alkylenexe2x80x9d linking groups in the definitions above are optionally substituted by one or more C1-6 alkyl;
with the proviso that R1, R2 and R3 are not all H;
hereinafter referred to as xe2x80x9csubstances of the inventionxe2x80x9d.
xe2x80x9cAlkylxe2x80x9d groups and the alkyl moiety of xe2x80x9calkoxyxe2x80x9d groups can be straight-chain, branched or cyclic where the number of carbon atoms allows.
xe2x80x9cHalogenxe2x80x9d means F, Cl, Br or I.
The two definitions given for the R4 moiety are of course tautomeric. The skilled man will realise that in certain circumstances one tautomer will prevail, and in other circumstances a mixture of tautomers will be present.
Pharmaceutically-acceptable salts are well know to those skilled in the art, and for example include those mentioned by Berge et al, in J. Pharm.Sci., 66, 1-19 (1977). Suitable acid addition salts are formed from acids which form non-toxic salts and include the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, hydrogenphosphate, acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, gluconate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate and p-toluenesulphonate salts.
When one or more of the substituents on the compound of formula (I) contains an acidic moiety, suitable pharmaceutically acceptable base addition salts can be formed from bases which form non-toxic salts and include the aluminium, calcium, lithium, magnesium, potassium, sodium, zinc, and pharmaceutically-active amines such as diethanolamine, salts.
The compounds of formula (I) having an acidic moiety can exist as one or more zwitterions. It is to be understood that all such zwitterions are included within the scope of the invention.
Certain of the compounds of the formula (I) may exist as geometric isomers. The compounds of the formula (I) may possess one or more asymmetric centres and so exist in two or more stereoisomeric forms. The present invention includes all the individual stereoisomers and geometric isomers of the substances of the invention and mixtures thereof.
Preferably R1 is H, CN, halogen or methyl optionally substituted by one or more halogen.
More preferably R1 is H, CN, Cl, Br or methyl.
Most preferably R1 is Cl or Br.
Preferably R2 is H, halogen, C1-6 alkyl optionally substituted by one or more halogen, aryl. CH2OR8. (Cn-alkylene)CONR5R6, CO2H or CH2NR5R6.
More preferably R2 is H, Cl, methyl, phenyl, CONHCH2Ph, CH2OPh, CH2NCH3Bn, or pyrrolidinomethyl.
Most preferably R2 is H.
Preferably R3 is H, Cl, Br, CF3, aryl, (Cn-alkylene)CO2H, (Cn-alkylene)CO2(C1-6 alkyl), (Cn-alkylene)CONR5R6, CHxe2x95x90CHR7, CHxe2x95x90CHCO2H, CHxe2x95x90CHCONR5R6, CHxe2x95x90CHSO2NR5R6, Cxe2x95x90CR7, O(Cm-alkylene)OH, O(Cm-alkylene)OR8, OR8, O(Cm-alkylene)CONR5R6, CH2OR8, or CH2NR5R6.
More preferably R3 is CHxe2x95x90CHCO2H, (2-carboxypyrrolidino)SO2CHxe2x95x90CH, (cyanophenyl)CHxe2x95x90CH, or (carboxyphenyl)CHxe2x95x90CH.
Yet more preferably R3 is CHxe2x95x90CHCO2H, (2-carboxypyrrolidino)SO2CHxe2x95x90CH, (3-cyanophenyl)CHxe2x95x90CH, or (3-carboxyphenyl)CHxe2x95x90CH.
Most preferably R3 is (2-carboxypyrrolidino)SO2CHxe2x95x90CH, (3-cyanophenyl)CHxe2x95x90CH, or (3-carboxyphenyl)CHxe2x95x90CH.
A preferable group of substances of the invention are those wherein R1 is H, CN, Cl, Br or methyl; R2 is H, Cl, methyl, phenyl, CONHCH2Ph, CH2OPh, CH2NCH3Bn, or pyrrolidinomethyl; and R3 is CHxe2x95x90CHCO2H, (2-carboxypyrrolidino)SO2CHxe2x95x90CH, (3-cyanophenyl)CHxe2x95x90CH, or (3-carboxyphenyl)CHxe2x95x90CH.
A yet more preferable group of substances of the invention are those in which R1 is Cl or Br; R2 is H; and R3 is (2-carboxypyrrolidino)SO2CHxe2x95x90CH, (3-cyanophenyl)CHxe2x95x90CH, or (3-carboxyphenyl)CHxe2x95x90CH.
A further preferred group of substances of the invention are those mentioned below in the Examples and the salts and solvates thereof.
Another aspect of the invention is a pharmaceutical composition comprising a substance of the invention according to the above definitions and a pharmaceutically-acceptable carrier.
Yet another aspect of the invention is a substance of the invention according to the above definitions for use as a medicament.
A further aspect of the invention is the use of a substance of the invention according to the above definitions, for the manufacture of a medicament for the treatment of a condition or process mediated by uPA, such as angiogenesis (neo-vascularization), bone restructuring, psoriasis, embryo implantation in the uterus, infiltration of immune cells into inflammatory sites, ovulation, spermatogenesis, tissue remodelling during wound repair and organ differentiation, fibrosis, local invasion of tumours into adjacent areas, metastatic spread of tumour cells from primary to secondary sites, and tissue destruction in arthritis.
Yet another aspect of the invention is a method of treatment of a condition or process mediated by uPA, such as angiogenesis (neo-vascularization), bone restructuring, psoriasis, embryo implantation in the uterus, infiltration of immune cells into inflammatory sites, ovulation, spermatogenesis, tissue remodelling during wound repair and organ differentiation, fibrosis, local invasion of tumours into adjacent areas, metastatic spread of tumour cells from primary to secondary sites, and tissue destruction in arthritis, comprising administering a therapeutic amount of a substance of the invention or composition according to the above definitions.
It is to be appreciated that reference to treatment includes prophylaxis as well as the alleviation of established detrimental symptoms of uPA-mediated conditions and processes, such that administration of the uPA inhibitor has a beneficial effect.
The substances of the invention may be separated and purified by conventional methods.
Separation of any diastereomeric mixtures may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of a compound of formula (I) or a suitable salt or derivative thereof. An individual enantiomer of a compound of formula (I) may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereomeric salts formed by reaction of the corresponding racemate with a suitably optically active acid or base.
The invention further provides Synthetic Methods for the production of substances of the invention, which are described below and in the Examples, in conjunction with the Preparations. The skilled man will appreciate that the substances of the invention could be made by methods other than those herein described, by adaptation of the methods herein described in the sections below and/or adaptation thereof, and of methods known in the art.
In the Synthetic Methods below, unless otherwise specified, the substituents are as defined above with reference to the compounds of formula (I) above.
Where desired or necessary the compound of formula (I) is converted into a pharmaceutically acceptable salt thereof. A pharmaceutically acceptable salt of a compound of formula (I) may be conveniently be prepared by mixing together solutions of a compound of formula (1) and the desired acid or base, as appropriate. The salt may be precipitated from solution and collected by filtration, or may be collected by other means such as by evaporation of the solvent.
Synthetic Methods
Method 1
Compounds of formula (I) can be obtained from the corresponding 2-aminopyridine derivative (II) by reaction with cyanamide (NH2CN) or a reagent which acts as a xe2x80x9cNHC+xe2x95x90NHxe2x80x9d synthon such as carboxamidine derivatives, e.g. 1H-pyrazole-1-carboxamidine (M. S. Bernatowicz, Y. Wu, G. R. Matsueda, J. Org. Chem., 1992, 57, 2497), the 3,5-dimethylpyrazole analogue thereof (M. A.Brimble et al, J. Chem.Soc.Perkin Trans.I (1990)31 1), simple O-alkylthiouronium salts or S-alkylisothiouronium salts such as O-methylisothiourea (F. El-Fehail et al, J.Med.Chem.(1986), 29, 984), S-methylisothiouronium sulphate (S.Botros et al, J.Med.Chem.(1986)29,874; P. S. Chauhan et al, Ind. J. Chem., 1993, 32B, 858) or S-ethylisothiouronium bromide (M. L. Pedersen et al, J.Org.Chem.(1993) 58, 6966). Alternatively aminoiminomethanesulphinic acid, or aminoiminomethanesulphonic acid may be used (A. E. Miller et al, Synthesis (1986) 777; K. Kim et al, Tet.Lett.(1988) 29,3183). 
Other methods for this transformation are known to those skilled in the art (see for example, xe2x80x9cComprehensive Organic Functional Group Transformationsxe2x80x9d, 1995, Pergamon Press, Vol 6 p639, T. L. Gilchrist (Ed.); Patai""s xe2x80x9cChemistry of Functional Groupsxe2x80x9dVol. 2. xe2x80x9cThe Chemistry of Amidines and Imidatesxe2x80x9d1991, 488).
2-Aminopyridines (II) may be prepared by standard published methods (see for example, xe2x80x9cThe Chemistry of Heterocyclic Compoundsxe2x80x9d Vol. 38 Pt. 2 John Wiley and Sons, Ed. F. G. Kathawala, G. M. Coppolq, H. F. Schuster) including, for example, by rearrangement from the corresponding carboxy-derivative (Hoffman, Curtius, Lossen, Schmidt-type rearrangements) and subsequent deprotection.
Alternatively, 2-aminopyridines may be prepared by direct displacement of a ring hydrogen using the Chichibabin reaction (A. F. Pozharskii et. al. Russian Chem. Reviews, 1978, 47, 1042. C. K. McGill et. al Advances in Heterocyclic Chemistry 1988, Vol. 44, 1)
2-Aminopyridines (II) may alternatively be prepared from the corresponding 2-halopyridines by direct displacement of a leaving group such as Cl or Br with a nitrogen nucleophile such as azide (followed by reduction), or by ammonia, or through Pd-catalysis with a suitable amine (such as benzylamine) followed by deprotection using standard conditions well-known in the art. Examples of such chemistry is outlined in xe2x80x9cThe Chemistry of Heterocyclic Compoundsxe2x80x9d Vol. 14, Pts. 2 and 3 John Wiley and Sons, in particular Pt. 2, (1961), Pt. 3 (1962), Pt. 2- supplement (1974) and Pt. 3-supplement (1974).
2-Halopyridines may be prepared by methods well known in the literature. For example, by treatment of 2-hydroxypyridines (2-pyrimidinones) with halogenating agents such as SOCI2 (Y. S. Lo. Et. Al. Syn. Comm., 1988, 19, 553), POCl3 (M. A. Walters, Syn. Comm., 1992, 22, 2829), or POBr3 (G. J. Quallich, J. Org. Chem., 1992, 57, 761). Alternatively, 2-alkoxypyridines may be transformed to the corresponding 2-aminopyridines under Vilsmeir-Haack conditions such as POCl3+DMF (L-L Lai et. Al. J Chem. Res. (S), 1996, 194). The corresponding N-oxide may be treated with suitable halogenating reactions to directly produce 2-halopyridinesxe2x80x94e.g. POCl3/PCl5 (M. A. Walters, Tetrahedron Lett., 1995, 42, 7575). Direct halogenation of the 2-position is possible in the presence of certain ring substituents (M. Tiecco et. al. Tetrahedron, 1986, 42, 1475, K. J. Edgar, J. Org Chem., 1990, 55, 5287).
Method 2
Compounds of formula (I) can be obtained from the corresponding 2-aminopyridine derivative (II) as defined in Method 1 above, via reaction with a reagent which acts as a protected amidine(2+) synthon (III): 
such as a compound PNHC(xe2x95x90Z)NHP1, PNxe2x95x90CZ1NHP1 or PNHCZ1xe2x95x90NP1, where Z is a group such as O. or S and Z1 is a leaving group such as Cl, Br, I, mesylate. tosylate, alkyloxy, etc., and where P and P1 may be the same or different and are N-protecting groups such as are well-known in the art, such as t-butoxycarbonyl, benzyloxycarbonyl, arylsulphonyl such as toluenesulphonyl, nitro, etc.
Examples of reagents that act as synthons (III) include N, Nxe2x80x2-protected-S-alkylthiouronium derivatives such as N, Nxe2x80x2-bis(t-butoxycarbonyl)-S-Me-isothiourea, N, Nxe2x80x2-bis(benzyloxycarbonyl)-S-methylisothiourea, or sulphonic acid derivatives of these (J. Org. Chem. 1986, 51, 1882), or S-arylthiouronium derivatives such as N, Nxe2x80x2-bis(t-butoxycarbonyl)-S-(2,4-dinitrobenzene) (S. G. Lammin, B. L. Pedgrift, A. J. Ratcliffe, Tet. Lett. 1996, 37, 6815), or mono-protected analogues such as [(4-methoxy-2,3,6-trimethylphenyl)sulphonyl]-carbamimidothioic acid methyl ester or the corresponding 2,2,5,7,8-pentamethylchroman-6-sulphonyl analogue (D. R. Kent, W. L. Cody, A. M. Doherty, Tet. Lett., 1996, 37, 8711), or S-methyl-N-nitroisothiourea (L. Fishbein et al, J.Am. Chem.Soc. (1954) 76, 1877) or various substituted thioureas such as N, Nxe2x80x2-bis(t-butoxycarbonyl)thiourea (C. Levallet, J. Lerpiniere, S. Y. Ko, Tet. 1997, 53, 5291) with or without the presence of a promoter such as a Mukaiyama""reagent (Yong, Y. F.; Kowalski, J. A.; Lipton, M. A. J. Org. Chem., 1997, 62, 1540), or copper, mercury or silver salts, particularly with mercury (II) chloride. Suitably N-protected O-alkylisoureas may also be used such as O-methyl-N-nitroisourea (N. Heyboer et al, Rec.Chim. Trav.Pays-Bas (1962)81,69). Alternatively other guanylation agents known to those skilled in the art such as 1-H-pyrazole-1-[N,Nxe2x80x2-bis(t-butoxycarbonyl)]carboxamidine, the corresponding bis-Cbz derivative (M. S. Bernatowicz, Y. Wu, G. R. Matsueda, Tet. Lett. 1993, 34, 3389) or mono-Boc or mono-Cbz derivatives may be used (B. Drake. Synthesis, 1994, 579, M. S. Bernatowicz.. Tet. Lett. 1993, 34, 3389). Similarly, 3,5-dimethyl-1-nitroguanylpyrazole may be used (T. Wakayima et al, Tet.Lett.(1986)29,2143).
The reaction can conveniently be carried out using a suitable solvent such as dichloromethane, N,N-dimethylformamide (DMF), methanol.
The reaction is also conveniently carried out by adding mercury (II) chloride to a mixture of the aminopyridine (II) and a thiourea derivative of type (III) in a suitable base/solvent mixture such as triethylamine/dichloromethane. 
The product of this reaction is the protected pyridinylguanidine (IV), which can conveniently be deprotected to give (I) or a salt thereof. For example, if the protecting group P and/or P1 is t-butoxycarbonyl, conveniently the deprotection is carried out using an acid such as trifluoroacetic acid (TFA) or hydrochloric acid, in a suitable solvent such as dichloromethane, to give a trifluoroacetate (triflate) salt of (I), either as the mono- or ditriflate.
If P and/or P1 is a hydrogenolysable group, such as benzyloxycarbonyl, the deprotection could be performed by hydrogenolysis.
Other protection / deprotection regimes include:
nitro (K. Suzuki et al, Chem.Pharm.Bull. (1985)33,1528, Nencioni et al, J.Med.Chem.(1991)34,3373, B. T. Golding et al, J.C.S.Chem.Comm.(1994)2613;
p-toluenesulphonyl (J. F. Callaghan et al, Tetrahedron (1993) 49 3479;
mesitylsulphonyl (Shiori et al, Chem.Pharm.Bull.(1987)35,2698, ibid.(1987)35,2561, ibid., (1989)37,3432, ibid., (1987)35,3880, ibid., (1987)35,1076;
2-adamantoyloxycarbonyl (Iuchi et al, ibid., (1987) 35, 4307; and
methylsulphonylethoxycarbonyl (Filippov et al, Syn.Lett.(1994)922)
It will be apparent to those skilled in the art that other protection and subsequent deprotection regimes during synthesis of a compound of the invention may be achieved by conventional techniques, for example as described in xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d by T W Greene and P G M Wuts, John Wiley and Sons Inc. (1991), and by P. J. Kocienski, in xe2x80x9cProtecting Groupsxe2x80x9d, Georg Thieme Verlag (1994).
Method 3
Compounds with the formula (I) can be obtained from compounds of formula (V): 
where Z is a suitable leaving group such as Cl, Br or OPh, by displacement of the leaving group by the free base of guanidine.
The free base of guanidine may conveniently be generated in situ from a suitable salt, such as the hydrochloride, carbonate, nitrate, or sulphate with a suitable base such as sodium hydride, potassium hydride, or another alkali metal base, preferably in a dry non-protic solvent such as tetrahydrofuran (TBF), DMSO, N,N-dimethylformamide (DMF), ethylene glycol dimethyl ether (DME), N,N-dimethyl acetamide (DMA), toluene or mixtures thereof. Alternatively it can be generated from a suitable salt using an alkoxide in an alcohol solvent such as potassium t-butoxide in t-butanol, or in a non-protic solvent as above.
The thus formed free guanidine can be combined with the compound of formula (V) and the reaction to form compounds of formula (I) can be carried out at from room temperature to 200xc2x0 C., preferably from about 50xc2x0 C. to 150xc2x0 C., preferably for between 4 hours and 6 days.
Method 4
Compounds of the formula (I) when one or more of R1-3 contains a hydroxy group, may be prepared from a suitably xe2x80x9cprotectedxe2x80x9d hydroxy derivative, i.e. a compound of the formula (I) where one or more of R1-3 contains a corresponding xe2x80x9cOP1xe2x80x9d where P2 is a suitable O-protecting group such as O-benzyl. The benzyl group may be removed for example by catalytic hydrogenation using a palladium on charcoal catalyst in a suitable solvent such as ethanol at about 20xc2x0 C. and elevated pressure, optionally in the presence of an excess of an acid such as HCl or AcOH, or TFA, or by other known deprotection methods.
Suitable O-protecting groups and protection/deprotection can be found in the texts by Greene and Wuts, and Kocienski, supra.
Method 5
Compounds of the invention where R2 or R3 is or contains a carboxylic acid group or carbamoyl group can be made from the corresponding compound where the substituent is or contains a nitrile by full or partial hydrolysis. Compounds of the invention where R2 or R3 is or contains a carboxylic acid group can be made from the corresponding compound where the substituent is a carbamoyl moiety, by hydrolysis. The hydrolysis can be carried out by methods well-known in the art, for example those mentioned in xe2x80x9cAdvanced Organic Chemistryxe2x80x9d by J. March, 3rd edition (Wiley-Interscience) chapter 6-5, and references therein. Conveniently the hydrolysis is carried out using concentrated hydrochloric acid, at elevated temperatures, and the product forms the hydrochloride salt.
Compounds of the formula (I) where one or more of R1, R2 or R3 is or contains Cl or Br may be dehalogenated to give the corresponding hydrido compounds of formula (I) by hydrogenolysis, suitably using a palladium on charcoal catalyst, in a suitable solvent such as ethanol at about 20xc2x0 C. and at elevated pressure.
Compounds of formula (I) in which one or more of R2 or R3 contains an amide moiety may be made via reaction of an optionally protected corresponding carboxy compound, by coupling with the amine of choice, e.g. via initial formation of the corresponding acid halide or mixed anhydride, and subsequent reaction with the amine, followed by deprotection if appropriate. Such transformations are well-known in the art.
Certain of the compounds of formula (I) which have an electrophilic group attached to an aromatic ring may be made by reaction of the corresponding hydrido compound with an electrophilic reagent. For example sulphonylation of the aromatic ring using standard reagents and methods, such as fuming sulphuric acid, gives a corresponding sulphonic acid. This can then be optionally converted into the corresponding sulphonamide by methods known in the art, for example by firstly converting to the acid chloride followed by reaction with an amine.
Certain of the substances of the invention can be made via cross-coupling techniques such as by reaction of a compound containing a bromo-substituent attached to e.g. an aromatic ring, with e.g. a boronic acid derivative, an olefin or a tin derivative by methods well-known in the art, for example by the methods described in certain of the Preparations below.
Certain of the substances of the invention having an electrophilic substituent can be made via halogen/metal exchange followed be reaction with an electrophilic reagent. For example a bromo-substituent may react with a lithiating reagent such as n-butyllithium and subsequently an electrophilic reagent such as CO2, an aldehyde or ketone, to give respectively an acid or an alcohol.
Substances of the invention are available by either the methods described herein in the Methods and Examples or suitable adaptation thereof using methods known in the art. It is to be understood that the synthetic transformation methods mentioned herein may be carried out in various different sequences in order that the desired compounds can be efficiently assembled. The skilled chemist will exercise his judgement and skill as to the most efficient sequence of reactions for synthesis of a given target compound.
It will be apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional techniques, for example as described in xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d by T W Greene and P G M Wuts, John Wiley and Sons Inc. (1999), and by P. J. Kocienski, in xe2x80x9cProtecting Groupsxe2x80x9dGeorg Thieme Verlag (1994).
For human use, the substances of the invention can be administered alone, but will generally be administered in admixture with a pharmaceutically acceptable diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, they can be administered orally, including sublingually, in the form of tablets containing such excipients as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents. They can be injected parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, they are best used in the form of a sterile aqueous solution or suspension which may contain other substances, for example, enough salt or glucose to make the solution isotonic with blood. They can be administered topically, in the form of sterile creams, gels, suspensions, lotions, ointments, dusting powders, sprays, drug-incorporated dressings or via a skin patch. For example they can be incorporated into a cream consisting of an aqueous or oily emulsion of polyethylene glycols or liquid paraffin, or they can be incorporated into an ointment consisting of a white wax soft paraffin base, or as hydrogel with cellulose or polyacrylate derivatives or other viscosity modifiers, or as a dry powder or liquid spray or aerosol with butane/propane, HFA or CFC propellants, or as a drug-incorporated dressing either as a tulle dressing, with white soft paraffin or polyethylene glycols impregnated gauze dressings or with hydrogel, hydrocolloid, alginate or film dressings. The compound or salt could also be administered intraocularly as an eye drop with appropriate buffers, viscosity modifiers (e.g. cellulose derivatives), preservatives (e.g. benzalkonium chloride (BZK)) and agents to adjust tenicity (e.g. sodium chloride). All such formulations may also contain appropriate stabilisers and preservatives. For oral and parenteral administration to human patients, the daily dosage level of the substances of the invention will be from 0.001 to 20, preferably from 0.01 to 20, more preferably from 0.1 to 10, and most preferably from 0.5 to 5 mg/kg (in single or divided doses). Thus tablets or capsules of the substances of the invention will contain from 0.1 to 500, preferably from 50 to 200, mg of active substance for administration singly or two or more at a time as appropriate.
The physician in any event will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case; there can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
It is to be appreciated that reference to treatment includes prophylaxis as well as the alleviation of established symptoms of the condition to be treated.
Test Methods
Substances were tested for their ability to inhibit human urokinase, human tPA and human plasmin, using substantially the same methods as described by Yang, et al, J.Med. Chem.,(1990)33,2961. The urokinase assay was carried out using S-2444 (Quadratech 820357) as substrate and the urokinase used was HMWT Human Urokinase (Calbiochem 672081). The tPA assay was carried out using S-2288 (Quadratech 820832) tPA substrate, Quadratech 321116 as tPA stimulator, and the tPA used was Human tPA (Quadratech 881157). The plasmin assay was carried out using human plasmin (Quadratech 810665) acting on Chromozym-PL (Boehringer 378461) as substrate.